The intraovarian site of 20 alpha-hydroxysteroid dehydrogenase activity (20 alpha-OH-SDH) was determined biochemically by measuring enzyme activity in homogenates of the whole ovary, or of isolated ovarian compartments, during the last third segment of pregnancy in the rat. In agreement, with previously reported histochemical evidence, an increase in 20 alpha-OH-SDH activity was observed in isolated corpora lutea, but not in the non-luteal compartment of the ovary. Enzyme activity in corpora lutea was low between days 16 and 22 of pregnancy, but increased markedly (4-6 fold) on day 23. Between days 17 and 20 of pregnancy, serum concentrations of progesterone declined from 130 +/- 3 to 80 +/- 3 ng/ml, while 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OH-P) concentrations declined from 34 +/- 3 to 18.5 +/- 3 ng/ml. Only later, between days 20 and 22 of pregnancy, was a significant decline in serum progesterone concentrations associated with an increase in serum 20 alpha-OH-P concentrations (50 +/- 15 ng/ml at 0800 h and 128.5 +/- 15 ng/ml at 1400 h on day 22). Thus the decline in progesterone concentration late in pregnancy can be explained only partially by conversion of progesterone to 20 alpha-OH-P. Further, a dissociation between changes in enzyme activity and in serum concentrations of 20 alpha-OH-P was also observed. A marked increase in serum levels of 20 alpha-OH-P on day 22 preceded any increase in enzyme activity by at least 10 h, and continued increases in enzyme activity on day 23 were not associated with any steady increase in peripheral 20 alpha-OH-P levels. We conclude from these observations that luteal regression is a more complex phenomenon than the regulation of a single enzyme, 20 alpha-hydroxysteroid dehydrogenase, and may involve regulation of both the synthesis and degradation of progesterone.